The adenine nucleotide ATP is released from erythrocytes (RBCs) into the circulation in response to hemoglobin (Hb) deoxygenation and cell deformation, and binds directly to purinergic receptors along the endothelium to evoke vasodilation and increase tissue blood flow and oxygen delivery. Isolated RBC ATP release during Hb deoxygenation, and plasma [ATP] and forearm blood flow (FBF) responses during exercise and systemic hypoxia, are impaired in healthy older vs. young adult humans. However, the underlying cause of these age-associated impairments remains unclear. Thus, the overall aim of this proposal is to identify changes within the RBC that contribute to impaired ATP release and vascular control with advancing age. Specific Aim 1 will test the hypothesis that age-associated changes in RBC structure and function - specifically, membrane deformability, oxidative stress, and cyclic AMP signaling - are mechanistically-linked to impaired ATP release during Hb deoxygenation in isolated RBCs from older adults. To test this, isolated RBC ATP release in response to normoxic (pO2 ~110-120mmHg) and hypoxic (pO2 ~15-25mmHg) stimuli will be quantified in RBCs from young and older adults using the luciferin-luciferase technique after incubation with either saline (control) or pharmacological agents to modify membrane deformability (Y-27632/ diamide), oxidative stress (ascorbic acid), or intracellular [cAMP] (cilostazol). Specific Aim 2 will test the hypothesis that in vivo treatment of impaired RBC ATP release with age will improve isolated RBC ATP release during Hb deoxygenation and in vivo vascular and plasma ATP responsiveness to systemic hypoxia and graded-intensity handgrip exercise in older adults, and that this will be associated with improved exercise capacity. To test this, the treatment from Specific Aim 1 that elicits the greatest improvement in Hb deoxygenation-induced ATP release in RBCs from older adults will be identified, and the corresponding pathway will be targeted using in vivo, systemic administration of either fasudil (deformability), ascorbic acid (oxidative stress), or cilostazol (cAMP signaling), in young and older adults. When peak plasma concentration of the drug is achieved, RBCs will be isolated and Hb deoxygenation-induced ATP release will be quantified as in Specific Aim 1; in conjunction, FBF responses to systemic isocapnic hypoxia (80% SpO2) and graded-intensity handgrip exercise (5%, 15%, 25% MVC) will be quantified using Doppler ultrasound and plasma [ATP] will be measured using the luciferin-luciferase technique. Submaximal rhythmic handgrip exercise time-to-fatigue will be assessed as an index of exercise capacity. The expected outcomes will provide novel insight into the impairments in RBC function and subsequent in vivo vascular control that occur with normal human aging; this insight is relevant to NHLBI's mission and research priorities, particularly those of the Vascular Biology and Hypertension Branch in the Division of Cardiovascular Sciences, given that impaired vascular control with advancing age leads to an increased risk of cardiovascular/ischemic disease and a decline in functional capacity and overall quality of life.